Low volume filtration column devices and methods of filtering therewith

ABSTRACT

This relates to filter columns for isolating nucleic acids, particularly at small elution volumes. The filter column is adapted for stable placement within the upper portion of standard plastic collection tubes of various sizes. The body of the filter column has a number of surfaces to accommodate placement within variously sized collection tubes. The filter column contains nucleic acid-specific filter which can be located at alternate regions within the filter column, providing different filter surface areas and loading volume capacities using the same column body. The filter column has an opening on an upper end adapted to be sealed by a cap. A method for recovering nucleic acids using such filter column is also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. Ser. No. 10/209,508,filed Jul. 30, 2002, which is a continuation-in-part of U.S. Ser. No.09/882,530, filed Jun. 15, 2001, the entire contents of both of thesepatent applications are hereby incorporated herein by reference as iffully set forth herein.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to filtration filter columns and methods offiltering therewith. More specifically, this invention relates to systemand methods for isolating nucleic acids such as ribonucleic acid (RNA)and deoxyribonucleic acid (DNA) from other materials such as enzymes,salts, buffers, small molecules, and cellular debris.

2. Discussion of the Related Art

Isolation and purification of nucleic acids play a central role inmodern molecular biology, and increasingly in medicine. Both laboratoryand diagnostic research require the use of nucleic acids in gene cloningand genetic analysis. Many of these techniques require keepingribonucleic acid (RNA) or deoxyribonucleic acid (DNA) pure and free ofcontamination. In many instances, the availability of small amounts ofstarting sample material poses a problem during isolation of the nucleicacid. The limited amount of sample material makes the need to limit lossof the sample material a critical concern.

A known method for isolating nucleic acids from a small amount ofstarting material includes the use of a spin filter column (“filtercolumn”) that contains a nucleic acid binding material (i.e., a filter).Examples of binding material/filters include silicas like glass powder,silica particles, glass microfibers, diatomaceous earth, etc. Thesefilters are often associated with a “filter surface area.” This filtersurface area is not limited to the surface area of a side of the filter.Instead, since the filters are usually comprised of microscopic fibers,particles, porous substances, etc., the filter surface area is actuallydefined by the surface area of the components, which comprise thefilter. For example, a filter that comprises glass microfibers may havea filter surface area defined by the surface area of the microfiberswithin the filter (either all of the microfibers or a portion thereof).

In some cases, filter columns may isolate nucleic acids directly fromcells or biological tissue. In the first step a filter column isinserted into a microcentrifuge tube (e.g., a 1.5 ml tube) and asolution containing nucleic acids along with undesirable impurities isloaded into the top of the filter column. Depending upon theapplication, the starting material containing the nucleic acids isprepared from cells that have been treated with a disrupting solutioncausing the release of the nucleic acids. Alternatively, the nucleicacid solution is the product of an earlier reaction step. In eithercase, the nucleic acid binds to the filter column filter in the presenceof a chaotropic agent. Then the filter column is centrifuged in amicrocentrifuge. Centrifugation forces the solution through the filtercolumn's filter and binds the nucleic acid to the filter. Next, thefilter with the nucleic acids bound therein is washed by applying awashing solution to the top of the filter column and centrifuging again.After each wash the filter column can be removed from the collectiontube so that the material in the collection tube may be removed.Finally, placement of an elution buffer (usually water having a specificpH) at the top of the column and applying centrifugation elutes thenucleic acid that is bound to the filter. Given the proper pH, thenucleic acid dissolves and elutes with the liquid into the collectiontube. It is important to note that the volumes of the binding and washsolutions can be relatively large, thus necessitating the use of alarger (1.5-2.0 mL) tube. On the other hand, the volumes for elution areoften smaller, making it desirable to have a smaller tube for theelution step. Eluting directly into a smaller tube allows one to proceedto the next reaction step in the small tube, rather than having topipette out of the large tube. It is desirable to use a small tube indownstream processes. As discussed herein, use of a pipette isundesirable as it introduces the risk of loss of samples as well ascontamination of the sample. Moreover, as described herein, there areadditional benefits in keeping the sample solution in a smaller tube.

Several companies provide kits that include filter columns designed touse this technique for isolating nucleic acids. QIAGEN, Promega, andBoehringer Mannheim GmbH offer filter columns based on theabove-described principle. However, existing filter columns cannot beused interchangeably with collection tubes of different sizes. Instead,these previously known filter columns only fit into a single sizecollection tube (e.g., a standard 1.5-2.0 mL microcentrifuge tube.)

This limitation presents a problem as many applications may benefit if asingle filter column could be interchanged with collection tubes ofmultiple sizes. For example, given a small amount of nucleic acid in thestarting material, it is best to elute the purified nucleic acid into avery small volume of fluid so that nucleic acid does not become toodilute in the fluid. Obviously, the resulting combination of elutionbuffer and nucleic acid will occupy a small volume. Many applicationsthat require processing of nucleic acid may benefit when storing thissmall volume of material in a smaller sized collection tube. Forinstance, an application such as amplification of the purified DNA bypolymerase chain reaction (PCR) requires placement of the nucleic acidinto a thin-walled 0.5 mL or 0.2 mL microcentrifuge tube.

Accordingly, it may seem ideal to use a smaller filter column, which isspecifically designed to fit a 0.5 mL or 0.2 mL microcentrifuge tube.However, a significant drawback is that these smaller filter columnslimit the amount of wash and binding solution that can be passed throughthe column with each bind/wash. This limitation necessitates additionalwash steps and increased handling of the filter column andmicrocentrifuge tube, thus presenting an undesirable increased risk ofcontamination. Also, as discussed above, the requirement of largevolumes of binding and wash solutions often necessitate the use of alarger collection tubes (e.g., a 1.5-2.0 mL microcentrifuge tube.)

The remaining alternative is to use a filter column specificallydesigned to fit into a larger collection tube (e.g., a 1.5 mL-2.0 mLmicrocentrifuge tube.) While this alternative minimizes the additionalwash steps and increased handling discussed above, the alternativepresents additional problems. For instance, after purification, thenucleic acid solution must be eluted from the filter column into anappropriately sized 1.5 mL-2.0 mL microcentrifuge tube. As discussedabove, many applications may benefit by storing the elution buffer andnucleic acid in a smaller sized microcentrifuge tube. Consequently, theelution buffer and nucleic acid must then be transferred (e.g., byaspiration) into a smaller (e.g. 0.5 mL) tube. Again, this extratransfer step introduces the undesirable potentials of contamination andloss of some of the nucleic acid.

The present invention involves the elution of purified nucleic acidsinto small volumes (generally less than 20 microliters). The filters ofthe filtration columns described are adapted to optimally function withsmall elution volumes by minimizing fluidic holdup during thepurification and elution procedures, and by tailoring the filtercharacteristics.

In view of the above, there remains a need to use different-sizedcollection tubes with a single filter column. The ability to usedifferent-sized collection tubes with a single filter column,particularly when dealing with small elution volumes, overcomes theproblems associated with the existing art.

The invention described herein addresses the problems discussed above.Moreover, the invention described herein allows centrifugation from onefilter column into at least two distinct sizes of collection tubes. Thedisclosed invention may be used with commercially available collectiontubes

BRIEF SUMMARY OF THE INVENTION

According to one aspect of the invention, there is provided a filtercolumn for isolating nucleic acids from a liquid sample. The filtercolumn includes a body having a passageway extending therethrough. Thebody has a first end, a second end, an outer surface, an inner surfaceand a longitudinal axis. The outer surface has at least one bearingsurface for seating on at least one collection tube. The inner surfacehas at least one angled surface located proximate or above the bearingsurface. The angled surface forms an angle with the longitudinal axisthat is less than approximately 90 degrees. A filter is disposed withinthe passageway.

According to another aspect of the invention, there is provided a filtercolumn that includes a body having a passageway extending therethrough.The body has an outer surface, an inner surface and a longitudinal axis.A filter is disposed within the passageway and the filter has aperimeter. A retainer is disposed within the passageway above thefilter. The retainer has an outer edge, an inner edge, and an uppersurface that is beveled.

According to another aspect of the invention, there is provided a filtercolumn that includes a body having a passageway extending therethrough.The body has an outer surface, an inner surface, and a longitudinalaxis. A filter is supported within the passageway and the filter has aperimeter. At least a portion of the filter proximate to the perimeteris compressed relative to a portion of the filter that is inward fromthe perimeter.

According to another aspect of the invention, there is provided a filtercolumn that includes a body having a passageway extending therethrough.The body has an outer surface, an inner surface, and a longitudinalaxis. A filter is disposed within the passageway and the filter has aperimeter. At least a portion of the filter has a greater densityrelative to another portion of the filter.

According to another aspect of the invention, there is provided a filtercolumn for isolating nucleic acids into solution of a preselectedelution volume. The filter column includes a body having a passagewayextending therethrough. The body of the filter column has a first end, asecond end, an outer surface and an inner surface. At least one filteris disposed within the passageway. The filter has a wetting capacitythat is approximately equal to the preselected elution volume.

According to another aspect of the invention, there is provided a filtercolumn for isolating nucleic acids into solution of a preselectedelution volume. The filter column includes a body having a passagewayextending therethrough. The body of the filter column has a first end, asecond end, an outer surface and an inner surface. At least one filteris disposed within the passageway. The filter has a wetting capacitythat is less than the preselected elution volume.

According to yet another aspect of the invention, there is provided amethod for isolating nucleic acid material. The method includes the stepof providing a filter column that is comprised of a body having apassageway extending therethrough. The body has an outer surface and aninner surface. The filter column also includes at least one filterdisposed within the passageway. The filter has a wetting capacity. Themethod also includes the step of providing a solution containing nucleicacid material. The solution containing nucleic acid is transferred tothe filter of the filter column. The solution is transferred from thefilter column. Elution buffer is added to the filter in an amountsubstantially equal to the wetting capacity of the filter. Nucleic acidis eluted from the filter column.

According to another aspect of the invention, there is provided a filtercolumn for isolating nucleic acids into solution of a preselectedelution volume. The filter column includes a body having a passagewayextending therethrough. The body of the filter column has a first end, asecond end, an outer surface and an inner surface. A filter is disposedwithin the passageway. The filter has a shape that defines a volume. Thefilter is configured such that the volume defined by the shape of thefilter is substantially equal to the preselected elution volume.

According to yet another aspect of the invention, there is provided amethod for isolating nucleic acid material. The method includes the stepof providing a filter column that is comprised of a body having apassageway extending therethrough. The body has an outer surface and aninner surface. The filter column also includes a filter disposed withinthe passageway. The filter has a shape that defines a volume. The methodalso includes the step of providing a solution containing nucleic acidmaterial. The solution containing nucleic acid is transferred to thefilter. The solution is transferred from the filter column. Elutionbuffer is added to the filter in an amount substantially equal to thevolume defined by the shape of the filter. Nucleic acid is eluted fromthe filter column.

According to yet another aspect of the invention, there is provided amethod for isolating nucleic acid material. The method includes the stepof providing a filter column that is comprised of a body having apassageway extending therethrough. The body has an outer surface and aninner surface. The filter column also includes a filter disposed withinthe passageway. The method also includes the step of providing asolution containing nucleic acid material. The filter column is locatedin fluid communication with a first collection vessel. The solutioncontaining nucleic acid is transferred to the filter. The solution istransferred from the filter column at a first speed followed by a secondspeed wherein the first speed is slower than the second speed. Elutionbuffer is added to the filter. Nucleic acid is eluted from the filtercolumn.

According to yet another aspect of the invention, there is provided amethod for isolating nucleic acid material. The method includes the stepof providing a filter column that is comprised of a body having apassageway extending therethrough. The body has an outer surface and aninner surface. The filter column also includes a filter disposed withinthe passageway. The method also includes the step of providing asolution containing nucleic acid material. The filter column is locatedin fluid communication with a first collection vessel. The solutioncontaining nucleic acid is transferred to the filter of the filtercolumn. The solution is transferred from the filter column. Elutionbuffer is added to the filter. Nucleic acid is eluted from the filtercolumn at a first speed followed by a second speed wherein the firstspeed is slower than the second speed.

According to another aspect of the invention there is provided a filtercolumn. The filter column includes a body having a passageway extendingtherethrough. The body has a first end, a second end, an outer surface,and an inner surface. The outer surface includes at least two bearingsurfaces for seating the filter column on collection vessels of at leasttwo different sizes. A filter is disposed within the passageway and thecross-sectional area of the passageway is substantially constant.

BRIEF DESCRIPTION OF THE DRAWINGS

A clear conception of the advantages and features constituting theinvention, and of the components and operation of model systems providedwith the invention, will become more readily apparent by referring tothe exemplary, and therefore nonlimiting, embodiments illustrated in thedrawings accompanying and forming a part of this specification, whereinlike reference numerals (if they occur in more than one view) designatethe same elements. Consequently, the claims are to be given the broadestinterpretation that is consistent with the specification and thedrawings. It should be noted that the features illustrated in thedrawings are not necessarily drawn to scale.

FIGS. 1A, 1B, 1C and 1D illustrate variations of filter columns of thepresent invention appropriate for mating with collection tubes ofvarying sizes.

FIG. 2A illustrates a sectional view of a variation according to theinvention. FIG. 2B illustrates a sectional view 200 of FIG. 2A.

FIGS. 3A and 3B show perspective and cross-sectional views,respectively, of a variation of the retaining ring according to theinvention. FIG. 3C shows an exploded view of the assembly of a filter,ring and support within the filter column of the invention.

FIG. 4 illustrates a cut-away perspective view of a variation of afilter column of the present invention containing a filter, ring andsupport.

FIG. 5A illustrates a cross-sectional view of a filter column of thepresent invention removably seated in a first collection tube. FIG. 5Billustrates a sectional view 500 of FIG. 5A.

FIG. 6 illustrates a cross-sectional view of the filter columnillustrated in FIG. 5 removably seated in a second collection tube beingof a different size than the first collection tube of FIG. 5.

FIGS. 7A and 7B illustrate additional aspects of the invention that maybe combined with any variation of the invention singly or incombination.

FIGS. 8, 9 and 10 illustrate a method of the present invention.

FIG. 11 illustrates a filter column of the present invention for usewith a carrier device.

FIG. 12 illustrates a perspective view of the filter column of thepresent invention having vents located in the lid.

DETAILED DESCRIPTION OF THE INVENTION

The following discussion of the variations of the invention and thereference to the attached drawings are for explanatory purposes and donot exhaustively represent the possible combinations and variations ofthe invention. Those skilled in the art will readily appreciate thatmany variations may be derived using the following description. Thefollowing examples are intended to convey certain principles of theinvention. These examples are not intended to limit the scope of theclaims to any particular example. It is understood that the claims areto be given their broadest reasonable interpretation in view of thedescription herein, any prior art, and the knowledge of those ofordinary skill in the field.

FIG. 1A shows a profile of a variation of the present invention. In thisvariation, the filter column 1 includes a passageway extendingtherethrough and a body portion that includes a first body portion 3, asecond body portion 5, and a third body portion 7. The filter column 1also contains a first bearing surface 9 and a second bearing surface 11located between body portions 3, 5, and 7 as illustrated. The bearingsurfaces are intended to permit placement of the filter column 1 invarious collection vessels or tubes of at least two different sizes(e.g., see FIGS. 5 and 6). Accordingly, the present inventioncontemplates that the bearing surfaces, either alone, or along with bodyportion(s) adjacent to a bearing surface, serve to provide stability ofa filter column when seated within a collection tube. Such stableplacement is necessary for the intended use of the filter column (e.g.,during centrifugation, vacuum filtering, handling, adding/removingmaterial, etc.) Furthermore, although bearing surfaces 9, 11 are locatedat the intersection of the respective body portions, in other variationsof the invention the bearing surfaces are located anywhere along thevarious body portions. As illustrated in FIG. 1B, the bearing surfaces10, 12 are located along respective body portions of a filter column 1.Additionally, while bearing surfaces 9, 11, are illustrated as beingtapered, the invention is not limited as such.

Variations of the invention also include filter columns with more thanthree body portions as well as filter columns with one or more bearingsurfaces. The variation of the spin-column depicted in FIG. 1 furtherincludes an outer rim 17 adjacent to a top 15 of the filter column 1.The outer rim 17 may also provide an additional bearing surface 19 andmay also aid in grasping and manipulating the filter column 1. This rimis also used for mating with upstream devices that transfer solution tothe invention such as an extraction device. The bottom of the filtercolumn may be reduced in diameter 13 to assist in retaining a filter(not shown) within the filter column 1.

Another variation is shown in FIGS. 1C and 1D. The filter column 100includes an inner surface 102 and an outer surface 104. A first bodyportion 103 and a second body portion 105 may also be defined. Thefilter column 100 includes at least one bearing surface 109 locatedbetween first body portion 103 and second body portion 105. A secondbearing surface 111 is located between the second body portion 105 and arim 117 adjacent to the top 115 of the filter column 100. As shown, thebearing surfaces 109 and 111 are formed in the outer surface 104. Theinner surface 102 does not have any angled surfaces. The inner surface104 forms a passageway 121 that extends throughout the length, from thebottom 113 to the top 115 of the filter column 100. The passageway 121has a cross-sectional area that is substantially constant throughout thelength of the passageway 121; however, the invention is not so limited.The inner passageway 121 may be tapered, include angled surfaces, andbasically have a cross-sectional area that is not constant along thelength of the passageway. As shown in FIG. 1C, the filter column 100 isformed in one piece.

FIG. 1D, illustrates a filter column 100 that is formed from twopieces-an inner portion 119 and an outer portion 120. The outer portion120 may be separable form the inner portion 119 in one variation.Together, the inner portion 119 and the outer portion 120 provide anouter surface 104 that includes at least one bearing surface. At leasttwo bearing surfaces are included such that the filter column is adaptedto seat on collection vessels of at least two different sizes. Thefilter column 100 of FIG. 1D includes a first body portion 103, a secondbody portion 105 and a third body portion 107. The filter column 100includes at least one bearing surface 109 located between the first bodyportion 103 and the third body portion 107. A second bearing surface 111is located between the second body portion 105 and the rim 117 adjacentto the top 115 of the filter column 100. A third bearing surface 121 islocated between the third body portion 107 and the second body portion105. Of course, any number of body portions and/or bearing surfaces maybe formed with the bearing surfaces being located anywhere along anybody portion.

It is noted that the body of the filter column may be adapted asrequired to accommodate any particular filtration process for example,centrifugation, vacuum filtering, or any known filtering process. Forexample, if a filter column of the present invention is intended for usewith vacuum filtering, the filter column may also include a manifold toaccommodate the vacuum. Such modifications are well known to thosefamiliar with filter columns and methods of using such devices.

The dimensions of the body portions 3, 5, 7, 103, 105, 107 are selectedso that the filter column 1 may fit into various collection tubes thatare sized for the respective body portion and bearing surface.Variations of the invention include sizing of a filter column to includebody portions and bearing surfaces that accommodate both a 1.5-2.0 mLand a 0.5 mL microcentrifuge tube. An example of such tubes includes PGCScientifics No. 16-8105-52 (1.5 mL) supplied by PGC ScientificsCorporation of Maryland, Eppendorf No. 22 36 430-8 (0.5 mL) supplied byEppendorf AG of Germany, Gene Amp No. N801-0611 (0.5 mL) supplied byPerkin-Elmer Life Science of Massachusetts, Ciro ManufacturingCorporation of Pompano, Fla. (2.0 ml) and MJ Research, Inc. of Waltham,Mass. (0.5 ml). For example, the opening diameter for a 1.5 mL-2.0 mLmicrocentrifuge tube may range from 0.32-0.37 in. The opening diameterfor a 0.5 mL microcentrifuge tube may range from 0.25-0.27 in. It isintended that, where appropriate, the definition of diameter of thefilter column may include any such external feature as crush ribs,buttress, collar, or any other feature that may be present on a portionof a filter column body that is intended for insertion into a collectiontube. The invention is not limited to filter columns or collection tubeshaving circular cross-sections.

While the invention may be suited for use with the tubes describedabove, the invention is not limited to compatibility with such tubes.Moreover, although variations of the inventive device described hereinare discussed for use with existing microcentrifuge tubes ranging insize from 0.5 mL capacity to 1.5-1.5 mL capacity, the invention is notlimited as such. Instead, the inventive device may be applied tocollection tubes as described herein, centrifuge tubes of any size, orany type of collection tube where a benefit from the improvements of thecurrent invention are desirable. Furthermore, the invention may beadapted to accommodate any number of combinations of large and smallcollection vessels, including, but not limited to a filter columnadapted to accommodate a large 0.5 mL tube and a small 0.2 mL tube, or afilter column adapted to accommodate a large 1.5 mL tube and a small 0.2mL tube.

The height of the filter columns of the present invention is selected sothat the filter column along with the particular collection tube usedwill fit within the centrifuge apparatus (e.g., an Eppendorf 5415Ccentrifuge supplied by Eppendorf AG of Germany.) For example, for a 0.5mL microcentrifuge tube to fit in the centrifuge previously listed, theheight which protrudes from the 0.5 mL microcentrifuge tube (i.e.,referring to FIG. 1A, the height from the top of bearing surface 11 tothe top of the device) must be below 0.625 in. preferably below 0.5 in.Of course, height is not a constraint when used with vacuum filtering.

The filter columns of the present invention may be fabricated frommaterials readily known to those familiar with existing filter columns.Such materials include, but are not limited to polypropylene,polycarbonate, polyethylene, polyethylene terephthalate, fluoropolymerssuch as polytetrafluoroethylene and polyvinylidine flouride, polyaryleneether ketones, co-polymers and any thermoplastic or other commonly usedmaterial. It is often desirable to use a material, which isthermoplastic to allow molding of the columns. The columns can also bemachined out of appropriate materials. In some cases, it is desirable tochoose materials for the filter column, which permit sterilizationand/or the removal of contaminants and harmful agents including theremoval of nucleic acids and nucleases, thereby, allowing the filtercolumn, filter, and sample to be nuclease-free.

FIG. 2 illustrates the internal body structure of a variation of afilter column of the present invention. In particular FIG. 2A,illustrates a cross-sectional view of a filter column 1 comprising abody having a passageway 21 extending therethrough. The body portionhaving an inner surface, an outer surface, a top end and a bottom end.In the variation shown in FIG. 2A, the filter column 1 includes a firstbody portion 3, a second body portion 5, and a third body portion 7. Thebody portions 3, 5, and 7 are interconnected in a telescoping fashionsuch that the width of the first body portion 3 is greater than thewidth of the second body portion 5 which is greater than the third bodyportion 7. As can be seen, body portions of different widths formbearing surfaces therebetween. The body portions together with thebearing surfaces enable the filter column to seat on collection vesselsof different sizes. Still referencing FIG. 2A, for example, the filtercolumn mates with a larger first collection vessel which seats againstthe bearing surface located between the first body portion 3 and thesecond body portion 5 such that the second body portion 5 is securelylocated inside the larger second collection vessel in a friction-fitengagement. A snap-fit engagement, or adhesive may also be employed tosecure the filter column to the collection vessel. The filter columnalso mates with a smaller second collection vessel which seats againstthe bearing surface located between the second body portion 5 and thethird body portion 7 such that the third body portion 7 is locatedinside the smaller first collection vessel in a friction-fit engagement.Of course, the filter column can mate with a relatively large collectionvessel which seats against the bearing surface located between the rimand first body portion 3 such that the first body portion 3 is securelylocated inside the collection vessel. The filter column may be securedto the collection vessel using any means known to one skilled in theart. Also, in one variation, the cross-sectional area of the passageway21 at the first body portion 3 is greater than the cross-sectional areaof the second body portion 5 which is greater than the cross-sectionalarea of the third body portion 7.

Liquid solutions may be loaded into the top of the filter column 15 anda lid may also be used. Typically, liquid solutions include thenucleotide-containing solution, wash or rinsing solutions and an elutionbuffer (e.g. water or tris/ethylenediamine-tetra-acetate (TE)). Thenucleic acid solution can be from a lysate (e.g. isolated directly fromcells) or nucleic acids from a reaction mixture. The nucleic acids fromreaction mixtures could be from reactions such as PCR, DNA or RNApolymerization, reverse transcription, etc. Before being loaded into thefilter column the nucleic acid solution is usually combined with abinding buffer containing a chaotropic agent to aid in binding thenucleic acid to the filter. The compositions of binding buffers, wash orrinsing solutions and elution buffers are well known in the field andcan be found in U.S. Pat. No. 5,075,430 to Little, U.S. Pat. No.5,808,041 to Padhye, et al., and U.S. Pat. No. 5,652,141 to Henco, etal., the entirety of each is hereby incorporated by reference herein.

The variation of the invention depicted in FIG. 2A also illustrates avent 29. The vent 29 may be located along an interior surface of apassageway 21 of a filter column 1 but will be placed in fluidcommunication with an exterior of the filter column 1. The vent 29permits venting of pressure within the passageway 21 during placement ofa lid (not shown) in the top opening 15 of the filter column 1. Withouta vent, the placement of a lid could increase pressure within thepassageway 21 such that sample material is forced out of the bottom 13of the filter column 1. Although such displacement of the material maynot have an effect on the function of a device of the present invention,such an occurrence may be undesirable. The device 1 may have any numberof vents. These vents may be placed randomly or spaced evenly apart on awall of a passageway. In one variation (not shown) four vents are placedat intervals of 90 degrees along the walls of the passageway. In anothervariation, a vent is placed in another location in the filter column 1or even within a lid itself as described with respect to FIG. 12. Inanother variation, no vents are employed.

FIG. 12 illustrates a perspective view of a filter column 130 accordingto the present invention wherein one or more vents 131 are located inthe lid 132. Vents 131 permit venting of air and prevent buffer, wash orelution fluid, for example, from being inadvertently pushed through thefilter when the lid 132 is closed down onto the column. As shown in FIG.12, the vent 131 has a curved surface 133. Also, the vent 131 is taperedsuch that the vent 131 is narrower towards the top 134 of the lid 132.These scalloped vents 131 that taper towards the top 134 of the lid 132provide a more robust air and fluid barrier when the lid 132 is closedpreventing fluid from spraying from, around, or through the lid 132.This variation allows no fluid to exit the column after the lid 132closes with a snap-fit bead or ridge 135 located on the passageway ofthe column 130. Also, this variation makes manufacture of the filtercolumn easier and less expensive because the injection molding tool doesnot have to accommodate a vent and a bead in the same location asrequired when the vent is formed in the passageway of the column.Therefore, the molding tool is easier to fabricate. Although four vents131 are shown in the lid 132, the invention is not so limited and anynumber of vents 131 can be included in the lid. Similarly, the inventionis not limited to scalloped or tapered vents.

FIG. 2B shows greater detail 200 of the juncture between two bodyportions. The wall 201 of the second body portion 5 connects to the wallof the third body portion 7 by an intermediary wall 204. Theintermediary wall 204 has an outer surface 206 and an inner surface 210.The outer surface 206 of this intermediary wall 204 forms a bearingsurface 11 as previously described. The inner surface 210 of thisintermediary wall 204 is angled with respect to body portions 5 and 7such that fluid in the filter column does not pool on the inner surface210 while the filter column is in a stationary vertical position orwhile the filter column is undergoing centrifugation. An angled surfacewithin the passageway need not be located at the intermediary wall. And,as shown above with respect to FIGS. 1C and 1D, an angled surface is nota necessary characteristic of the invention, as internal components,such as the filter and filter support, as well as external mating partscan be supported and/or retained by other methods such as with adhesive.

A funnel angle for the angled surface 210 is defined as the anglebetween the longitudinal axis A of the filter column and the angledsurface 210 in question. The funnel angle of the angled surface is lessthan 90 degrees. In another variation, not only the juncture 200 of bodyportions 5 and 7 form a funnel angle that is less than 90 degrees, butalso, the funnel angle of all junctures of the filter column passagewaythrough which fluid must flow is less than 90 degrees whether or not theangled surface is located at the intermediary wall. This designadvantageously prevents the creation of a shelf that is a potentialsource of fluid holdup when force is applied to the filter column. Ifthe funnel angle of any angled surface is 90 degrees or more, fluid canbe trapped on the angled surface. The funnel angle of a surfaceindicates whether fluid will collect on the surface. Fluid holdup shouldbe avoided because it reduces the efficacy of the filter columnpurification. Thus, the inner passageway is designed to prevent fluidholdup within the filter column.

FIG. 2B shows the funnel angle 202 for the inner surface 210 when thedirection of force applied is parallel to the wall 201 of the secondbody portion 5. As shown, the funnel angle 202 is approximately 45degrees. This angle is less than 90 degrees required to prevent fluidholdup on this surface. FIG. 2B represents the case, of a filter columnadapted for spinning in a microcentrifuge with a swinging-arm rotor sothat the centrifugal force applied is substantially parallel to thewalls of the second body portion. Alternatively, it would also apply ifa vacuum or gravity were used to apply force to an upright filtercolumn. Generally, the primary adaptation of the filter column is for itto be employed in a microcentrifuge with a rotor that holds the columnat an angle of 45 degrees with respect to the direction of centrifugalforce, and the depicted embodiment is designed for that case.

Preventing fluid holdup is particularly critical when eluting from thefilter column into a small volume (e.g., less than 20 microliters). Manymicrocentrifuges have fixed-angle rotors where the tubes are held at aforty-five degree angle. In this example, the angle between the angledinner surface 210 and the wall 201 of the second body portion 5 is lessthan 45 degrees, resulting in a funnel angle that is less than 90degrees. For other microcentrifuges that do not hold the tubes at 45degrees, the surfaces of the passageway through the filter column couldbe designed so that the funnel angle remains less than 90 degreesthroughout the passageway of the filter column. In another variation,the funnel angle 202 is approximately 30 degrees at one or morejunctures.

As illustrated in FIG. 2A, the filter column 1 of the present inventionalso allows for varying placement of a filter (not shown) within apassageway 21 of the filter column 1. For example, placement of a filterin a bottom 23 of the second body portion 5 permits the filter column 1to provide a certain volume capacity. The volume capacity of the filtercolumn 1 can increase by changing the location of the filter to a bottom25 of the third body portion 7. Accordingly, the same filter column 1body may be used for varying application and more than one filter may bedisposed within the filter column. To maximize the volume of fluid thatcan be added to a filter column of the present invention, a filter canbe located in the lowest chamber. Moreover, locating the filter towardsa middle chamber reduces the volume of fluid that may be added butpermits a larger filter surface area as the diameter of the middlechamber may be greater than the diameter of a lower chamber. For thefilter, it is desirable to have a high surface area glass fiber filter(e.g., a borosilicate glass). The filter may be one that is adapted toisolate nucleic acids from a liquid sample by, for example,centrifugation, vacuum filtering, or any other filtering method. Surfacearea refers to the total surface area of all the fibers and not just thearea of the disk. Moreover, filter columns of the present invention mayhave a microliter capacity greater than 200 microliters. Anothervariation of the filter column having a microliter capacity rangingbetween 50 microliters and 1000 microliters. The microliter capacity isdefined by the volume within a passageway of the filter column that isabove the filter and not occupied by other components such as a lid.

The filter chosen can be silica or other types such as polymericmembranes, and may also contain other functional groups for purificationof the nucleic acid such as ion exchange groups or groups which wouldspecifically bind nucleic acid sequences. The structure and thickness ofthe filter will determine the filter wetting capacity. Wetting capacityis the amount of solution that can occupy the filter. For example, afilter made of glass fibers will have a wetting capacity approximatelydefined by the volume of space between the glass fibers. Thus, a roughapproximation of the wetting capacity of a filter made of glass fibercould be determined by subtracting the volume of glass fiber from thetotal volume of the filter. The volume of the glass fiber can bedetermined by dividing the mass of the glass fiber filter by thespecific gravity of the glass fiber. The wetting capacity can bedetermined by empirically measuring the amount of fluid a volume offilter material can hold. The wetting capacity is also called the voidvolume and other ways of measuring the void volume can be used todetermine the wetting capacity. The quantitative definition of maximumwetting capacity is the void space within the filter. The void spacewithin the filter can be calculated by taking the total space, bothsolid and void, that is circumscribed by the outermost fibers of thefilter minus the solid space occupied by the filter fibers themselves.The space occupied by the filter fibers can be calculated by dividingthe mass of the filter by the specific gravity of the fiber material.Other techniques known to one skilled in the art could also be used todetermine wetting capacity and is within the scope of the presentinvention.

Wetting capacity can be used to optimize the dimensions of the filterused in the filter column, particularly for low-volume purification. Amismatch between the wetting volume and the volume of solution appliedto the filter can decrease the yield of recovery of purification using afilter. For example, eluting purified nucleic acid into a preselectedsmall volume (20 mL or less) of elution buffer when the wetting capacityof the filter is greater than the elution volume could result inincomplete recovery of the purified nucleic acid material. Thus, theelution buffer will not contact all parts of the filter that have boundnucleic acid, and therefore not all of the bound nucleic acid will beeluted. Additionally, if the elution volume is much larger than thewetting capacity, the effective in-filter residence time of the fluid isdecreased from 100%, down to the ratio of wetted fluid to total volume.It is desirable to substantially match the wetting capacity of thefilter chosen to the elution volume that is selected for the processesto be performed. To recover purified material into a low-volume ofelution buffer (e.g., 20 microliters) using the inventive filter column,the filter has a similarly low wetting capacity. In one variation, thewetting capacity of the filter is substantially equal to the volume ofelution buffer that is employed. A precise match between elution volumeand wetting capacity is not required as long as it is plus or minus 10%of the elution volume. In one variation, the filter is configured suchthat the volume, that includes the space of both void and solid fiber asdefined by the shape of the filter, is substantially equal to thepreselected elution volume. In yet another variation, the wettingcapacity is between approximately 8 microliters and 12 microliters.

In another variation, the wetting capacity is slightly less than thevolume of elution buffer employed. In another variation the wettingcapacity is approximately 66 percent to 100 percent of the elutionvolume. In another variation, the wetting capacity is approximately 50percent to 100 percent of the elution volume. The wetting capacity ofthe filter column is easily modified by changing the dimensions of thefilter, e.g. filter thickness, diameter, etc. If the filter iscompressed, the wetting capacity is the resulting wetting capacity ofthe compressed filter.

At low volumes it may also be difficult to completely wet the filterbecause the surface tension may inhibit absorption into the filter. Thisis particularly critical when using low volumes of elution buffer. Onesolution is to include surface tension reducing agents, such asdetergents, in the elution buffer. For example, addition of 0.1% of thedetergent Triton X-100 could be included in the elution buffer to helpwet the filter when eluting purified nucleic acid.

Securing of the filter may be accomplished, for example, by placement ofa disk of porous substrate material (not shown) at approximatelyposition 25 of FIG. 2A. A filter membrane (not shown) is placed on topof the substrate material. Optionally, a retainer (not shown) is addedsuch that there is an interference or adhesion or interlocking betweenthe retainer and the wall of the passageway. Accordingly, the retainersecures the filter on the porous substrate. Another means of securingthe filter is to mold an integral grating within the passageway to seatthe filter. FIG. 3C illustrates placement of a filter using the poroussubstrate and a retainer in the shape of a ring.

FIG. 3A shows a perspective view of one embodiment of the retainer 33 ofthe present invention. The retainer 33 is shaped to substantiallyconform to the shape of the passageway. The retaining ring 33 includesan inner edge 302 and an outer edge 304. The inner edge 302 of theretaining ring 33 is beveled. This beveling creates a funnel angle thatreduces fluid holdup. FIG. 3B shows a side cross-sectional view of theretaining ring 33 of FIG. 3A. The outer edge of the filter 304 is notbeveled, and is designed to fit snugly against the walls of thepassageway of the filter column. Alternately, the outer edge 304 mayloosely fit and be attached to the inner wall of the passageway using anadhesive or other attachment means. As shown in FIG. 3B, the inner edge302 includes a lower surface 306 and an upper surface 308. Both thelower 306 and upper 308 surfaces of the inner edge 302 of the retainingring 33 are beveled in FIG. 3B such that the thickness of the retainingring 33 decreases with distance from the outer edge towards the inneredge. The angle as well as the extent of this beveling can be variedresulting in a different funnel angle for the upper surface 308 of theretaining ring, or a different compression profile of the filter fromthe lower portion of the retaining ring. In one variation, the entireupper surface 308 of the retaining ring 33 is beveled, eliminating anyshelf-like protrusion on which solution could collect while stationaryor while undergoing centrifugation or vacuum filtering. The lowersurface 306 may also have a similar beveled profile. In yet anothervariation, the funnel angle of the upper surface 308 is less than 90degrees. Also, either one of the lower or upper surfaces may be beveled.

FIG. 3C shows how the retaining ring 33 inserts into the column filterin order to secure the filter in place. FIG. 3C illustrates an explodedview of a retaining ring 33, filter 29 and filter support 31 fittinginto one embodiment of the filter column 1. The filter 29 is sandwichedbetween the retaining ring 33 and the filter support 31, and all threefit into the filter column. The filter support 31 and the retaining ring33 match the diameter of the portion of the filter column into whichthey will be inserted. In one variation, when the retaining ring 33 isinserted, it abuts the filter 29 acting to compress at least a portionof the filter 29 that is proximate to the outer perimeter relative to aportion of the filter that is inward from the perimeter. In anothervariation, the insertion of the retainer 33 acts to increase the densityof at least a portion of the filter relative to another portion of thefilter. If a retainer 33 as shown in FIG. 3 is employed, the at least aportion of the filter of greater density is proximate to the perimeterof the filter relative to a portion of the filter that is inward fromthe perimeter. In one case, the top surface 28 of the filter 29 adopts asurface profile that is substantially complementary to the surfaceprofile of the lower surface 306 of the retaining ring 33. A beveledlower surface 306 of the retaining ring 33 results in a gradient ofcompression across the filter 29 such that the middle region of thefilter is uncompressed or only slightly compressed relative to the edgeregions of the filter that are more compressed. The compression of thefilter decreases the available surface area and wetting capacity in thatregion. While not bound by theory, this compression is believed toincrease capillary affinity at the compressed circumference of thefilter, and therefore, help draw fluid into the filter and toward theedges. A retainer need not be employed to compress at least a portion ofthe filter. FIG. 4 shows a filter column with the filter secured.

FIG. 4 illustrates a cut-away profile view of a variation of a filtercolumn 1 of the present invention. The illustration shows a filter 29held in the lower portion of the filter column 1. In this variation, thefilter 29 is held between a porous support membrane 31, which has thesame diameter as the interior of the lower portion of the spin column 1.In this variation, the support membrane 31 is retained in place by theshoulder formed by the reduced diameter of the bottom 13 of the device.The filter 29 may be fixed against the filter support membrane 31 by aretaining ring 33, which fits securely against the inner wall of thelower portion of the filter column 1. The present invention is notlimited to the previous illustration. It will be apparent to thoseskilled with previously known filter columns to provide other means ofretaining the filter within a filter column. An example of a filter foruse with the invention includes a borosilicate glass fiber filter. Thedevice may also contain filters of other types such as polymericmembranes, and may also contain other functional groups for purificationof the nucleic acid such as ion exchange groups or groups which wouldspecifically bind nucleic acid sequences.

The porous substrate could be comprised of a material such as a sinteredpolyethylene or polypropylene as supplied by Porex of Fairburn, Ga. orGenPore of Reading, Pa. In some variations of the invention it isdesirable that the porous substrate is comprised of a hydrophobicmaterial in order to minimize holdup of the aqueous solution and toprevent fluid intended for residence in the filter from exiting thefilter prematurely. It was found that pore sizes in the range of 1micron to 150 microns are useful. When the filter column is used forvery low volumes of solution (e.g. elution buffer) it is preferred thatthe surface of the filter support that contacts the filter besubstantially smooth and free from excessive pits or cavities. Althoughthe filter support must be porous, gaps in the interface between thefilter support and the filter create regions where fluid can leave thefilter, reducing the amount of contact between the fluid and the filterfibers. Generally, it is better to have a more uniform and thereforemore substantially smooth surface for the filter support. A smalleraverage pore size usually results in a more uniform filter supportsurface. The primary function of the porous material is support for thefilter. It is also possible to have the porous support as integral tothe device. The porous support can also be in the form of an opengrating. It is also possible to have a filter that is strong enough tosupport itself or is materially combined with support material.

FIG. 5A illustrates a variation of a filter column 1 of the presentinvention that is mated with a standard collection tube 35 of a firstsize (e.g., a 1.5 mL tube.) Standard collection tubes contain anintegral body made of polymers such as polypropylene and have a standardinner diameter. As shown in FIG. 5A, the second body portion 5 of thefilter column 1 may fit securely against an inner wall of the collectiontube 35. The first bearing surface 9 may rest against a shoulder formedby a rim 37 of the collection tube 35. Accordingly, when the tube 35 andfilter column 1 spin in the centrifuge, liquid placed into the filtercolumn 1 is forced through a filter 29 and collects in the collectiontube 35. In this variation, the sizing of the first body portion 5 alongwith the bearing surface 9 permits stable placement of the filter column1 within in the collection tube 35. After centrifugation, the filtercolumn can be easily removed from the used collection tube, and placedinto a new collection tube or placed back into the original tube.

FIG. 5B illustrates a view of section 500 of FIG. 5A. A retaining ring33, filter 29 and a filter support 31 are disposed within a body portion501 of the filter column 1. The body portion 501 has a volume. Thefilter 29 is located between the filter support 31 and the retainingring 33. The top surface 28 of the filter conforms to the lower surface306 of the retaining ring 33 such that the outer portion 502 of thefilter 29 is more compressed relative to the inner portion 504 of thefilter 29. As can be seen in FIG. 5B, the compression profile of thefilter corresponds to the surface profile of the lower surface 306 ofthe retaining ring 33. In one variation, the filter has a compressionprofile such that the compression of the filter gradually increases withdistance towards the outer edge. The compressed filter defines a volume.In one variation, the compressed wetting capacity of the filter 29 isselected to be approximately equal to or slightly greater than theelution volume.

FIG. 6 illustrates the filter column 1 of FIG. 5 placed within acollection tube 39 (e.g., a 0.5 mL tube) that is smaller than the tubeillustrated in FIG. 5. As illustrated in FIG. 6, the third body portion7 of the filter column 1 fits securely into the inner walls of thecollection tube 39. During centrifugation, a bearing surface 11 of thefilter column 1 seats against a rim 41 of the collection tube 39.

FIG. 7A shows another variation of a filter column 42 of the presentinvention having features, which may be applied to any variation of theinvention disclosed herein. In this variation the filter column 42includes a lid 45 having a portion 47 which may be removably securedwithin a first end 49 of the filter column 42. The lid 45 may assist inpreventing contamination of the filter column 42 and any solutionsloaded therein and may provide a surface for labeling. As shown, thisvariation of the filter column 42 contains a vent 27. Although thisvariation depicts the lid 45 as being integral with the filter column 42via a hinge 51, the invention is not limited as such. For example, thepresent invention also includes a lid as being discrete from a filtercolumn.

As illustrated in FIG. 7A, the variation of the inventive filter columnalso may include one or more snap-fit beads or ridges 53 to assist inretention of a lid 45. In this variation the ridge 53 is included in thein a portion of a passageway in a first body portion 3 of the filtercolumn 42. As depicted in FIG. 7A, the lid 45 and hinge 51 may beattached to the underside of an outer rim 55 (e.g., subflush to the rim55) of the filter column 42. This placement allows the outer rim 55 tobe mated to other devices as required (e.g., a ExtracSure.™. sampleextraction device useful for laser capture microdissection supplied byArcturus Engineering of Mountain View, Calif. or other sample carrier).

Another aspect of the invention depicted in FIG. 7A is deformable ribs57, which may be located along an outside surface of the filter column42. The deformable ribs 57 assist in securing the filter column 42 in aslightly larger diameter tube (e.g., a 2.0 mL tube) by increasing adiameter of the filter column 42 for a friction-fit engagement, forexample. Accordingly, these ribs deform upon insertion of the filtercolumn 42 into a tube having a diameter slightly larger than thecorresponding body portion upon which the ribs 57 are situated. Thenumber and design of the deformation ribs 57 may vary as needed,however, the ribs should be placed on a portion of the filter columnthat accommodates the varying sizes of tubes. The invention alsocontemplates deformation ribs 57, which are either plastically orelastically deformable, or exhibit a limited degree of plastic orelastic deformation.

FIG. 7B illustrates another aspect of the invention, in which a filtercolumn 59 contains at least one protrusion 61, which extends radiallyaway from the filter column 59. Such protrusions 61 may serve as “fingergrips” to increase the ease with which the filter column 59 may bemanipulated.

FIGS. 8-10 represent a flow diagram illustrating isolation of nucleicacids such as DNA or RNA from a biological material 80 using a filtercolumn 86 of the present invention. FIG. 8 represents the first step ofplacing a lysate 82 of biological material 80 in a collection tube 84.This lysate 82 is spun to remove large lytic debris, and the “cleared”lysate is then applied to the top of a filter column 86 of the presentinvention.

Alternatively, the nucleic acid containing solution does not have tocome directly from lysis of biological materials. This method can alsopurify nucleic acids following nucleic acid amplification, enzymaticrestriction digestion, ligation, extension and virtually any solutioncontaining significant nucleic acids.

Where small amounts of nucleic acid solutions are used, it is desirableto pre-wet the filter column with binding buffer before applying thenucleic acid solution. Alternatively, or additionally, the solutioncontaining nucleic acid is transferred to the filter (wherein thenucleic acid material binds to the filter). This transfer may beperformed at a first speed or rate employing any method known to oneskilled in the art such as centrifugation or via vacuum. This bindingstep also includes a second step, which is performed at a high speed orrate of centrifugation, for example. Hence, solution is transferred fromthe filter column at a first speed followed by a second speed whereinthe first speed is slower than the second speed. In one variation inwhich centrifugation is employed, the first slow speed is approximately500 g or less, 1000 g or less, 1500 g or less, or 2000 g or less. Thisfirst step at a slow speed advantageously increases the resident timethat the solution containing the nucleic acid material is in contactwith the filter. The second relatively fast speed is approximately10,000 g or more.

FIG. 9A illustrates the solution passing though a filter of the filtercolumn 86 after centrifugation, during which the nucleic acids bind tothe filter. The flow-through solution (from which the nucleotides havebeen mostly extracted) is retained at the bottom of a collection tube88. Generally, tubes 88, 84 are discarded after use. Upon removal of thefilter column 86 from the tube 88, the binding buffer (flow through) isdiscarded, and the filter column is replaced in a collection vessel(either a new tube or the same tube). FIG. 9B represents repeatedwashing of the filter column 86 with a washing buffer and centrifugingafter each addition of washing solution into the top of the filtercolumn. Once again the flow-through accumulated at the bottom of thetube 88 is discarded. FIG. 10 represents the last step of transferringthe filter column 86 of the present invention into a smaller 0.5 mL tube92 and the addition of a small volume of elution buffer into the filtercolumn 86. Elution buffer is added to the filter in an amountsubstantially equal to the wetting capacity of the filter. Additionalelution buffer may also be added to ensure that filter is notunder-wetted. In one variation, additional elution buffer is added tothe filter column in an amount that is between zero and approximatelyfifty percent of the wetting capacity of the filter. Alternatively,additional elution volume is added in an amount that is between zero andapproximately 66% of the wetting capacity of the filter. In anothervariation, a volume of elution buffer is added to the filter column inan amount substantially equal to the volume defined by the shape of thefilter. The volume defined by the shape of the filter includes the spaceoccupied by both the void and solid filter fibers in a spacecircumscribed by the outermost fibers of the filter or approximately thevolume of fluid that can occupy the space bounded by the bottom surfaceof the filter, the top surface of the filter, and the edges of thefilter and/or the filter column walls. As a result of the addition ofthe elution buffer, the nucleic acids release from the filter into theelution buffer, which is centrifuged into the bottom of the 0.5 mL tube92. When small volumes of elution buffer (e.g., 20 microliters or less)are used, the step of eluting the material is performed in two parts.First, the elution step is performed at a low-speed followed by a secondstep having a slow speed relative to the first slow-speed step. Theelution step may be performed employing any method known to one skilledin the art including centrifugation or via vacuum. Hence, nucleic acidmaterial is eluted from the filter column at a first speed followed by asecond speed wherein the first speed is slower than the second speed. Ifcentrifugation is employed, the first slow speed is approximately 500 gor less, 1000 g or less, 1500 g or less, or 2000 g or less. The firstslow-speed step advantageously ensures that the filter is completelybathed in the elution buffer. A second spin at a higher speed (relativeto the first spin) of approximately 10,000 g or more is done to allowcollection of the maximum amount of elution buffer.

As shown in FIGS. 8-10, the inventive filter column permits use of morethan one collection tube. All of the centrifugation steps can take placein a standard bench-top microcentrifuge (for example, Eppendorf 5415C),usually at accelerations less than 20,000 g.

The biological material used with the filter column may also be providedby laser capture microdissection extraction device as described in U.S.patent application Ser. No. 09/844,187, entitled “LASER CAPTURE MICRODISSECTION (LCM) EXTRACTION DEVICE AND DEVICE CARRIER, AND METHOD FORPOST-LCM FLUID PROCESSING,” the entirety of which is hereby incorporatedby reference. As shown in FIG. 11, a filter column 1 attached to a LCMextraction device flange interface 94 which seats a laser capturemicrodissection extraction device 96. The biological material will belocated on a bottom surface of the extraction device 96.

While the present invention has been described with reference to one ormore particular variations, those skilled in the art will recognize thatmany changes may be made thereto without departing from the spirit andscope of the present invention. Each of these embodiments and obviousvarious thereof are contemplated as falling within the spirit and scopeof the claimed invention, which is set forth in the claims.

1. A filter column 1, comprising: a body having a passageway extendingthrough the body; the body having a first end, a second end, an outersurface and an inner surface; a filter located in the passageway; a lidfor closing one end of the passageway; and at least one vent located inthe lid, wherein the vent(s) is tapered.
 2. The filter column of claim 1wherein the vent(s) is tapered such that the vent is narrower towardsthe top of the lid.
 3. The filter column of claim 1 wherein the vent(s)prevents fluid from being pushed through the filter when the lid isclosed down onto the column.
 4. The filter column of claim 1 wherein thevent(s) prevents fluid from escaping from around or through the lid. 5.The filter column of claim 1 wherein the vent(s) prevents fluid frombeing pushed through the filter when the lid is closed down onto thecolumn and prevents fluid from escaping from around or through the lid.6. A filter column comprising: a body having a passageway extendingthrough the body; the body having a first end, a second end, an outersurface and an inner surface; a filter located in the passageway; a lidfor closing one end of the passageway; at least one vent located in thelid; and at least one bearing surface on an outer surface for seating onat least one collection tube.
 7. The filter column of claim 6 where theouter surface further includes a second bearing surface for seating onat least a second collection tube that has a different volume from theat least one collection tube.
 8. The filter column of claim 6 furtherincluding at least a first body portion and a second body portion;wherein the cross-sectional area of the passageway at the first bodyportion is greater than the cross-sectional area of the passageway atthe second body portion.
 9. The filter column of claim 8 wherein thefilter is located in the second body portion.
 10. The filter column ofclaim 1 wherein the lid is connected to the body with a hinge.
 11. Thefilter column of claim 1 wherein the vent(s) includes a curved surface.